PhenolExtractionofrRNA(Ratliver)

LEVELII

Materials

• Ratliver(fastedrat)
• Liquidnitrogen
• p-Amino-salicicacid
• Phenolmixture
• Homogenizerorblender6
• Refrigeratedpreparativecentrifuge
• NaCl
• 95%and70%(v/v)ethanol

Procedure

1. Obtainaratwhichhasbeenfastedfor24hours(toremoveglycogenfromtheliver),decaptitate,exsanguinateandremovetheliverasrapidlyaspossIBLe.

2. Weightheliver,beingcarefulnottoallowtheittodehydrate.

3. Immediatelydroptheliverintoacontainerofliquidnitrogen.

   CAUTION:Liquidnitrogenwillcauseseverefrostbite!

4. Usingtheweightoftheliverasanindicationofthevolume(1gmofliverequivalentto1ml),add15volumesoffreshlyprepared6%para-amino-salicylate(pAS)toachilledblenderorhomogenizer.

5. Addanequalvolume(equaltothepAS)ofphenolmixturetotheblenderandturnontheblenderforashortbursttomixthepASandphenol.

   CAUTION:Phenolisextremelycaustic.

   Phenolcausessevereskinburns,yetitisalocalanesthetic.Youwillbeunawareoftheburnatfirst,exceptfortell-talediscolorationoftheskinandblisters.Youwillbecomeawareoftheburnastheanestheticpropertieswearoff.PhenolalsoreADIlydissolvesmostcountertopsandallrubbercompounds.

   CLEANUPALLSPILLSIMMEDIATELY!NOTIFYYOURINSTRUCTOROFANYSPILLSAFTERYOUHAVETHOROUGHLYRINSEDANDWASHEDAWAYANYMATERIALSINCONTACTWITHYOURSKIN.

6. Stoptheblenderandaddthefrozenliver(handletheliverwithlongforceps,ortongs).Blendtheentiremixture(pAS,phenolandliver)for30secondsatfullspeed.DonotblendforlongerperiodsoryouwillsheertheRNA.

7. Carefullytransferthehomogenatetoabeakerandcontinuetostirthemixturefor10minutesatroomtemperature.

8. Transferthehomogenatetonalgenecentrifugetubesandcentrifugethemixtureat15,600xgat4°Cfor20minutes.

9. Removethecentrifugetubesandcarefullyseparatetheupperaqueouslayerfromthelowerphenollayer.Takecarethatnoneofthewhiteinterphasematerialismixedintotheaqueouslayer.Theupperlayercanmostefficientlyberemovedbyusingalargehypodermicequippedwithalong,largebore,squaretippedneedle.Shouldsomeoftheinterphasematerialbestirredintotheaqueousphase,itwillbenecessarytorepeatstep8.

10. Measurethevolumeoftheaqueouslayeranddiscardthephenollayerandinterphasematerial.

11. Add3.0gramsofNaClper100ml.ofaqueousphaseandstiruntildissolved.

12. Add0.5volumesofphenolmixturetotheaqueousphase,placeintoasuitableflaskandshakevigorouslyforaboutfiveminutes.Recentrifugeasinstep8above,butfor10minutes.

13. Separatetheaqueousphaseandadd2.3volumesofcold95%ethanol.Allowthemixturetostandinthefreezeruntilaprecipitateforms.

14. CollecttheRNAprecipitatebycentrifugation,washoncein70%ethanolandstorein70%ethanolat0-5°C.

Notes

KnowledgeoftranscriptionisbasedonourABIlitytoextract"native"orfunctionalRNAmoleculesfromcells,withsubsequentuseofthosemolecules"invitro."Oneoftheearliestmethodsforthistypeofanalysisisaphenol-detergentextractionofRNA7coupledwithseparationofthevarioussizedmoleculesofRNAwithcentrifugationinagradient.

Thisbasicprocedureremainsusefultoday,althoughtherehavebeenmyriadadditionsandalterationstotheprocedureusingahostofextractiontechniquesandseparationprocedures(suchaselectrophoresisorcolumnchromatography).

Forthepurposesofintroductiontothetechnique,thisexerciseextractsRNAfromratliverusingaphenolextractionwhichyieldspredominantlyrRNAandtRNA.ThereissomemRNApresent,butitisvariableandshouldbeconsideredasabackgroundcontaminant.ThereisalsoagoodportionofsRNAcausedbysheeringoftheRNAduringhomogenization,andbyenzymaticdigestionbyRNAaseduringtheextraction.

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